Marjorie Safran

Environmental Iodine Intake and Thyroid Dysfunction During Chronic Amiodarone Therapy

Amiodarone, an iodine-containing drug used frequently in the treatment of cardiac arrhythmias and angina pectoris, has many effects on thyroid hormone metabolism, including decreasing the production of triiodothyronine (T3) and decreasing the clearance of thyroxine and reverse T3. These effects result in elevated serum thyroxine and reverse T3 concentrations and decreased serum T3 concentrations. In addition, iodine-induced hyperthyroidism or hypothyroidism may occur in patients chronically treated with amiodarone. This study is a retrospective analysis of the incidence of thyroid dysfunction in Lucca and Pisa, West Tuscany, Italy, and in Worcester, Massachusetts. Hyperthyroidism was a more frequent (9.6%) complication of amiodarone therapy in West Tuscany, where iodine intake is moderately low; hypothyroidism was more frequent (22%) in Worcester, where iodine intake is sufficient. In patients receiving chronic amiodarone therapy, clinically suspected hyperthyroidism is best confirmed by showing elevations in serum T3 or free T3 concentrations; hypothyroidism is best diagnosed by showing an elevated serum thyrotrophin concentration. Thyroid function should be carefully monitored in patients receiving amiodarone chronically, especially if they have goiter or Hashimotos thyroiditis.

The Role of Cell Cycle Regulatory Protein, Cyclin D1, in the Progression of Thyroid Cancer

Cell cycle progression is facilitated by cyclin-dependent kinases that are activated by cyclins including cyclin D1 and inactivated by cyclin-dependent kinase inhibitors (CDKIs) such as p27. Our previous studies have demonstrated decreased p27 expression in both papillary and more aggressive carcinomas of the thyroid compared to thyroid adenoma and almost similar level of cyclin D1 expression between thyroid adenoma and papillary carcinoma. These results indicate that CDKIs may have an important role in the carcinogenesis of the thyroid and that they probably have a limited role in malignant progression of the thyroid cancer. The role of cyclin D1 in malignant progression of thyroid carcinoma has yet to be established. We studied the expression of cyclin D1 by immunohistochemistry in 34 cases of conventional papillary carcinoma (CPC), 10 cases of minimally invasive follicular carcinoma (MIFC), and 32 cases of more aggressive thyroid carcinoma (ATC), which included 11 tall cell variants, one columnar cell variant of papillary carcinoma, seven insular carcinomas, and 13 anaplastic carcinomas. Cyclin D1 staining was classified by staining score as 0, negative; 1+, less than 25%; 2+, 25 to 50%; and 3+, more than 50% tumor cells staining positive. Kruskal-Wallis one-way ANOVA and Wilcoxon Rank Sum/Mann-Whitney U Test was used to assess the difference in the expression of cyclin D1 between the study groups. Twenty-eight out of the 34 CPCs were cyclin D1 positive, 24 (70%) were 1+, 3 (9%) were 2+, and one (3%) were 3+ positive. Seven of 10 MIFCs were cyclin D1 positive, five (71%) were 1+, and the remaining two (29%) were 2+ positive. On the other hand, 28 of 32 ATCs showed cyclin D1 immunostaining. Of these, three (9%) were 1+, five (13%) were 2+, and 20 (63%) were 3+ positive. This study demonstrates a significant overexpression of cyclin D1 in ATC compared CPC (P < .001) and MIFC (P < .005), suggesting that the cyclin D1 expression may play a role in tumor progression and may have prognostic significance in thyroid cancer.

AMIODARONE IODINE‐INDUCED HYPOTHYROIDISM: RISK FACTORS AND FOLLOW‐UP IN 28 CASES

Amiodarone, an iodine‐rich drug widely used for the treatment of cardiac tachyarrhythmias, may induce either hyperthyroidism or hypothyroidism. Of 467 patients chronically treated with this drug referred to our institution, amiodarone iodine‐induced hypothyroidism (AIIH) developed in 28 patients (6%). AIIH patients were subdivided into two groups according to the presence (group A) or absence (group B) of underlying thyroid abnormalities. Thyroid autoantibodies were present in 10 of 19 patients from group A and 0 of 9 patients from group B. The thyroid 24‐h radioiodine uptake (RAIU) was evaluated in 15 patients: low values (<4%) were found in three patients and detectable values (7–50%) were observed in 12. Perchlorate discharge tests were positive in all four patients tested. Follow‐up data were available in 20 patients (16 in group A and four in group B). Hypothyroidism was transient in 12 (60%) and persistent for several months after amiodarone withdrawal in eight (40%). While all patients in group B had transient hypothyroidism, 50% of patients with underlying thyroid abnormalities (group A) had persistent hypothyroidism. Thyroid autoantibodies were found in seven of eight patients with persistent hypothyroidism and in only three of 12 patients with transient hypothyroidism. Conversely, seven of 10 patients with positive thyroid autoantibodies had persistent hypothyroidism and 9 of 10 patients with undetectable thyroid autoantibodies had transient hypothyroidism. These data indicate that: (i) AIIH may develop in patients with or without underlying thyroid abnormalities; (ii) RAIU is inappropriately elevated in many patients with AIIH; (iii) intrathyroidal iodine is not organified; (iv) serum thyroid autoantibodies represent a risk factor for the development of AIIH; (v) AIIH spontaneously remits after amiodarone withdrawal in patients without thyroid abnormalities, but may persist in patients with concomitant thyroid disorders, especially those with circulating thyroid autoantibodies.

Follicular variant of papillary thyroid carcinoma: a comparative study of histopathologic features and cytology results in 141 patients.

OBJECTIVE To characterize the histopathologic features of follicular variant of papillary thyroid carcinoma (FVPC) and its cytology results on fine-needle aspiration (FNA) biopsy and compare them with those of papillary thyroid carcinoma (PC). METHODS We searched the University of Massachusetts Medical Center pathology database for all surgical specimens associated with a diagnosis of FVPC or PC between January 1992 and February 1998 and reviewed the related pathology reports. In addition, the associated preoperative FNA results were analyzed. RESULTS On initial assessment, FVPC was associated with a significantly lower incidence of cervical lymph node metastatic involvement in comparison with PC (5.6% versus 35.7%; P<0.001). Even though the mean size of FVPC was larger than that of PC (2.57 cm versus 1.75 cm; P<0.05), FVPC showed a lower incidence of thyroid capsule invasion (5.6% versus 11.4%), infiltrative resection margins (2.8% versus 20.0%; P = 0.01), local soft tissue invasion (7.0% versus 25.7%; P<0.005), and multicentricity (25.4% versus 47.1%; P<0.01). Lymphocytic thyroiditis was a common feature of both FVPC (36.6%) and PC (35.7%). FNA biopsy revealed the presence of malignant cells in 9.8% of patients with FVPC in comparison with 67.5% of patients with PC. Most cytology specimens of FVPC (58.8%) were interpreted as suspicious for a malignant lesion or as a follicular neoplasm. CONCLUSION FVPC is associated with a significantly lower incidence of cervical lymph node metastatic lesions and invasive histologic features than is PC. Long-term prospective clinical studies are needed to determine whether these findings translate into a more benign natural history for this variant of PC. Results of FNA biopsy in FVPC are more commonly interpreted as suspicious rather than malignant; this factor has major implications for preoperative planning.

Five patients with iodine-induced hyperthyroidism

Iodine-induced hyperthyroidism has been frequently described when iodine is introduced into an iodine-deficient area. However, it may also occur in patients with and without previous thyroid disease residing in iodine-sufficient areas. Five patients with iodine-induced hyperthyroidism seen in a 12-month period are described. All were exposed to iodine in the form of commonly used drugs (Betadine, Iodo-Niacin, amiodarone, and radiographic contrast dyes). The cause of iodine-induced hyperthyroidism is unclear, but it is probably more common in patients with goiters containing previously existing areas of autonomous function or iodine-poor thyroglobulin. Iodine-induced hyperthyroidism usually abates after iodine withdrawal in patients with multinodular goiters or normal thyroid glands. The hyperthyroidism is usually treated with beta-blockers and antithyroid thionamide drugs, although reinstitution of iodine to block thyroid hormone release or corticosteroids occasionally may be necessary. Iodine-containing drugs should be given with caution to patients with underlying thyroid disease.

Expression of Eukaryotic Translation Initiation Factors 4E and 2α Correlates with the Progression of Thyroid Carcinoma

Cell growth and proliferation depend on protein synthesis that is regulated, in part, by two eukaryotic translation initiation factors, eIF-4E and eIF-2alpha. These factors are transiently increased as normal cells respond to growth factors and are constitutively elevated in transformed cells. In cultured cells, eIF-4E facilitates cell cycle progression by increasing the expression of cell cycle promoting proteins including cyclin D1. Our previous study revealed elevated cyclin D1 expression in histologically more aggressive thyroid carcinomas as compared to conventional papillary carcinoma. We hypothesized that the increased cyclin D1 expression might correlate with increased eIF-4E expression. We, therefore studied the expression of eIF-4E by immunohistochemistry in 25 cases of conventional papillary carcinoma (CPC) and 28 cases of aggressive thyroid carcinomas (ATC), the latter included 11 tall cell/columnar cell variant of papillary carcinoma, 5 insular carcinomas, and 12 anaplastic carcinomas. We also analyzed the expression of eIF-2a in the same samples as this factor is usually regulated similarly to eIF-4E in cell culture models. Of the 25 CPC, 13 were eIF-4E positive (11 weakly and 2 strongly), and 19 were eIF-2a positive (14 weakly and 5 strongly). Conversely, of the 28 ATC, 25 were eIF-4E positive (4 weakly and 21 strongly), and 23 were eIF-2alpha positive (4 weakly and 19 strongly). There was a significantly increased expression of both eIF-4E (p < 0.001) and eIF-2alpha (p < 0.001) in ATC compared to CPC, suggesting that these translation initiation factors may play a role in the progression of thyroid cancer.

Cloning, Expression, and Functional Characterization of the Substrate Binding Subunit of Rat Type II Iodothyronine 5′-Deiodinase

Type II iodothyronine 5′-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this ∼200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an ∼60-kDa, cAMP-induced activation protein, and one or more uniden- tified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5′-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5′-deiodinase from a λzapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an ∼30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5′-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29GFP virus particles into the cerebral cortex of neonatal rats leads to a ∼2-fold increase in brain type II 5′-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5′-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

Catalytic Activity of Type II Iodothyronine 5′-Deiodinase Polypeptide Is Dependent upon a Cyclic AMP Activation Factor

Type II iodothyronine 5′-deiodinase is an ∼200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3′-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5′-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5′-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of ∼60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5′-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.

Effects of Amiodarone and Desethylamiodarone on Pituitary Deiodinase Activity and Thyrotropin Secretion in the Rat

The effect of acute administration of amiodarone, its major metabolite desethylamiodarone and iodine in an amount equal to that contained in amiodarone on serum thyroid hormone and thyrotropin (TSH) concentrations and hepatic and pituitary 5′ deiodination of thyroxine (T4) in the euthyroid and hypothyroid rat was evaluated. Amiodarone, desethylamiodarone and iodine all caused a decrease in serum T4 and triiodothyronine (T3) concentrations in euthyroid rats, while serum TSH concentrations and pituitary and hepatic 5′ deiodinase activities were decreased only in the amiodarone and desethylamiodarone-treated animals. Serum TSH was increased in the iodine treated rats. Amiodarone, but not iodine, decreased serum T3 and TSH concentrations and pituitary and hepatic 5′ deiodinase activities in hypothyroid rats. Inhibition of hepatic 5′ deiodinase activity was also observed by the addition of amiodarone in vitro in the absence of dithiothreitol (DTT) but not in the presence of DTT. The decrease in the serum T4 concentration observed with amiodarone and desethylamiodarone administration is probably secondary to the inhibitory effect of iodine released from the drugs on thyroidal T4 synthesis and secretion. Iodine inhibition of thyroidal T3 synthesis and secretion, decreased T4 substrate for a peripheral generation of T3 and inhibition of T4 to T3 conversion all contribute to the decrease in serum T3 observed. The decrease in the serum TSH concentration, despite low serum T4 and T3 concentrations and inhibition of pituitary 5′ deiodinase, suggest that amiodarone may function as a thyroid hormone agonist in the pituitary. Finally, the observation that amiodarone inhibits hepatic 5′ deiodination in vitro allows further evaluation of its mechanism of action.

Degradation and Recycling of the Substrate-binding Subunit of Type II Iodothyronine 5′-Deiodinase in Astrocytes

Thyroxine dynamically regulates levels of type II iodothyronine 5′-deiodinase (5′D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5′D-II is an ∼200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5′D-II. We show that T4 accelerates the rate of 5′D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5′D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane ∼2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5′D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways.